Production of peptides in plants as viral coat protein fusions

ABSTRACT

The present invention relates to foreign peptide sequences fused to recombinant plant viral structural proteins and a method of their production. Fusion proteins are economically synthesized in plants at high levels by biologically contained tobamoviruses. The fusion proteins of the invention have many uses. Such uses include use as antigens for inducing the production of antibodies having desired binding properties, e.g., protective antibodies, or for use as vaccine antigens for the induction of protective immunity, including immunity against parasitic infections.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation application of U.S.patent application Ser. No. 08/324,003, filed Oct. 14, 1994, which is acontinuation-in-part of U.S. patent application Ser. No. 08/176,414,filed on Dec. 29, 1993, and which is a continuation-in-part of U.S.patent application Ser. No. 07/997,733, filed Dec. 30, 1992.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of geneticallyengineered peptide production in plants, more specifically, theinvention relates to the use of tobamovirus vectors to express fusionproteins.

BACKGROUND OF THE INVENTION

[0003] Peptides are a diverse class of molecules having a variety ofimportant chemical and biological properties. Some examples include;hormones, cytokines, immunoregulators, peptide-based enzyme inhibitors,vaccine antigens, adhesions, receptor binding domains, enzyme inhibitorsand the like. The cost of chemical synthesis limits the potentialapplications of synthetic peptides for many useful purposes such aslarge scale therapeutic drug or vaccine synthesis. There is a need forinexpensive and rapid synthesis of milligram and larger quantities ofnaturally-occurring polypeptides. Towards this goal many animal andbacterial viruses have been successfully used as peptide carriers.

[0004] The safe and inexpensive culture of plants provides an improvedalternative host for the cost-effective production of such peptides.During the last decade, considerable progress has been made inexpressing foreign genes in plants. Foreign proteins are now routinelyproduced in many plant species for modification of the plant or forproduction of proteins for use after extraction. Animal proteins havebeen effectively produced in plants (reviewed in Krebbers et al., 1992).

[0005] Vectors for the genetic manipulation of plants have been derivedfrom several naturally occurring plant viruses, including TMV (tobaccomosaic virus). TMV is the type member of the tobamovirus group. TMV hasstraight tubular virions of approximately 300×18 nm with a 4 nm-diameterhollow canal, consisting of approximately 2000 units of a single capsidprotein wound helically around a single RNA molecule. Virion particlesare 95% protein and 5% RNA by weight. The genome of TMV is composed of asingle-stranded RNA of 6395 nucleotides containing five large ORFs.Expression of each gene is regulated independently. The virion RNAserves as the messenger RNA (mRNA) for the 5′ genes, encoding the 126kDa replicase subunit and the overlapping 183 kDa replicase subunit thatis produced by read through of an amber stop codon approximately 5% ofthe time. Expression of the internal genes is controlled by differentpromoters on the minus-sense RNA that direct synthesis of 3′-coterminalsubgenomic mRNAs which are produced during replication (FIG. 1). Adetailed description of tobamovirus gene expression and life cycle canbe found, among other places, in Dawson and Lehto, Advances in VirusResearch 38:307-342 (1991). It is of interest to provide new andimproved vectors for the genetic manipulation of plants.

[0006] For production of specific proteins, transient expression offoreign genes in plants using virus-based vectors has severaladvantages. Products of plant viruses are among the highest producedproteins in plants. Often a viral gene product is the major proteinproduced in plant cells during virus replication. Many viruses are ableto quickly move from an initial infection site to almost all cells ofthe plant. Because of these reasons, plant viruses have been developedinto efficient transient expression vectors for foreign genes in plants.Viruses of multicellular plants are relatively small, probably due tothe size limitation in the pathways that allow viruses to move toadjacent cells in the systemic infection of entire plants. Most plantviruses have single-stranded RNA genomes of less than 10 kb. Geneticallyaltered plant viruses provide one efficient means of transfecting plantswith genes coding for peptide carrier fusions.

SUMMARY OF THE INVENTION

[0007] The present invention provides recombinant plant viruses thatexpress fusion proteins that are formed by fusions between a plan viralcoat protein and protein of interest. By infecting plant cells with therecombinant plant viruses of the invention, relatively large quantitiesof the protein of interest may be produced in the form of a fusionprotein. The fusion protein encoded by the recombinant plant virus mayhave any of a variety of forms. The protein of interest may be fused tothe amino terminus of the viral coat protein or the protein of interestmay be fused to the carboxyl terminus of the viral coat protein. Inother embodiments of the invention, the protein of interest may be fusedinternally to a coat protein. The viral coat fusion protein may have oneor more properties of the protein of interest. The recombinant coatfusion protein may be used as an antigen for antibody development or toinduce a protective immune response.

[0008] Another aspect of the invention is to provide polynucleotidesencoding the genomes of the subject recombinant plant viruses. Anotheraspect of the invention is to provide the coat fusion proteins encodedby the subject recombinant plant viruses. Yet another embodiment of theinvention is to provide plant cells that have been infected by therecombinant plant viruses of the invention.

BRIEF DESCRIPTION OF THE FIGURES

[0009]FIG. 1. Tobamovirus Gene Expression

[0010] The gene expression of tobamoviruses is diagrammed.

[0011]FIG. 2. Plasmid Map of the TMV Transcription Vector pSNC004

[0012] The infectious RNA genome of the U1 strain of TMV is synthesizedby T7 RNA polymerase in vitro from pSNC004 linearized with KpnI.

[0013]FIG. 3. Diagram of Plasmid Constructions

[0014] Each step in the construction of plasmid DNAs encoding variousviral epitope fusion vectors discussed in the examples is diagrammed.

[0015]FIG. 4. Monoclonal Antibody (NVS3) Binding to TMV291

[0016] The reactivity of NVS3 to the malaria epitope present in TMV291is measured in a standard ELISA.

[0017]FIG. 5. Monoclonal Antibody (NYS1) Binding to TMV261

[0018] The reactivity of NYS1 to the malaria epitope present in TMV261is measured in a standard ELISA.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0019] Definitions and Abbreviations

[0020] TMV: Tobacco mosaic tobamovirus

[0021] TMVCP: Tobacco mosaic tobamovirus coat protein

[0022] Viral Particles: High molecular weight aggregates of viralstructural proteins with or without genomic nucleic acids

[0023] Virion: An infectious viral particle.

[0024] The Invention

[0025] The subject invention provides novel recombinant plant virusesthat code for the expression of fusion proteins that consist of a fusionbetween a plant viral coat protein and a protein of interest. Therecombinant plant viruses of the invention provide for systemicexpression of the fusion protein, by systemically infecting cells in aplant. Thus by employing the recombinant plant viruses of the invention,large quantities of a protein of interest may be produced.

[0026] The fusion proteins of the invention comprise two portions: (i) aplant viral coat protein and (ii) a protein of interest. The plant viralcoat protein portion may be derived from the same plant viral coatprotein that serves a coat protein for the virus from which the genomeof the expression vector is primarily derived, i.e., the coat protein isnative with respect to the recombinant viral genome. Alternatively, thecoat protein portion of the fusion protein may be heterologous, i.e.,non-native, with respect to the recombinant viral genome. In a preferredembodiment of the invention, the 17.5 KDa coat protein of tobacco mosaicvirus is used in conjunction with a tobacco mosaic virus derived vector.The protein of interest portion of the fusion protein for expression mayconsist of a peptide of virtually any amino acid sequence, provided thatthe protein of interest does not significantly interfere with (1) theability to bind to a receptor molecule, including antibodies and T cellreceptor (2) the ability to bind to the active site of an enzyme (3) theability to induce an immune response, (4) hormonal activity, (5)immunoregulatory activity, and (6) metal chelating activity. The proteinof interest portion of the subject fusion proteins may also possessadditional chemical or biological properties that have not beenenumerated. Protein of interest portions of the subject fusion proteinshaving the desired properties may be obtained by employing all or partof the amino acid residue sequence of a protein known to have thedesired properties. For example, the amino acid sequence of hepatitis Bsurface antigen may be used as a protein of interest portion of a fusionprotein invention so as to produce a fusion protein that has antigenicproperties similar to hepatitis B surface antigen. Detailed structuraland functional information about many proteins of interest are wellknown, this information may be used by the person of ordinary skill inthe art so as to provide for coat fusion proteins having the desiredproperties of the protein of interest. The protein of interest portionof the subject fusion proteins may vary in size from one amino acidresidue to over several hundred amino acid residues, preferably thesequence of interest portion of the subject fusion protein is less than100 amino acid residues in size, more preferably, the sequence ofinterest portion is less than 50 amino acid residues in length. It willbe appreciated by those of ordinary skill in the art that, in someembodiments of the invention, the protein of interest portion may needto be longer than 100 amino acid residues in order to maintain thedesired properties. Preferably, the size of the protein of interestportion of the fusion proteins of the invention is minimized (butretains the desired biological/chemical properties), when possible.

[0027] While the protein of interest portion of fusion proteins of theinvention may be derived from any of the variety of proteins, proteinsfor use as antigens are particularly preferred. For example, the fusionprotein, or a portion thereof, may be injected into a mammal, along withsuitable adjutants, so as to produce an immune response directed againstthe protein of interest portion of the fusion protein. The immuneresponse against the protein of interest portion of the fusion proteinhas numerous uses, such uses include, protection against infection, andthe generation of antibodies useful in immunoassays.

[0028] The location (or locations) in the fusion protein of theinvention where the viral coat protein portion is joined to the proteinof interest is referred to herein as the fusion joint. A given fusionprotein may have one or two fusion joints. The fusion joint may belocated at the carboxyl terminus of the coat protein portion of thefusion protein (joined at the amino terminus of the protein of interestportion). The fusion joint may be located at the amino terminus of thecoat protein portion of the fusion protein (joined to the carboxylterminus of the protein of interest). In other embodiments of theinvention, the fusion protein may have two fusion joints. In thosefusion proteins having two fusion joints, the protein of interest islocated internal with respect to the carboxyl and amino terminal aminoacid residues of the coat protein portion of the fusion protein, i.e.,an internal fusion protein. Internal fusion proteins may comprise anentire plant virus coat protein amino acid residue sequence (or aportion thereof) that is “interrupted” by a protein of interest, i.e.,the amino terminal segment of the coat protein portion is joined at afusion joint to the amino terminal amino acid residue of the protein ofinterest and the carboxyl terminal segment of the coat protein is joinedat a fusion joint to the amino terminal acid residue of the protein ofinterest.

[0029] When the coat fusion protein for expression is an internal fusionprotein, the fusion joints may be located at a variety of sites within acoat protein. Suitable sites for the fusion joints may be determinedeither through routine systematic variation of the fusion jointlocations so as to obtain an internal fusion protein with the desiredproperties. Suitable sites for the fusion jointly may also be determinedby analysis of the three dimensional structure of the coat protein so asto determine sites for “insertion” of the protein of interest that donot significantly interfere with the structural and biological functionsof the coat protein portion of the fusion protein. Detailed threedimensional structures of plant viral coat proteins and theirorientation in the virus have been determined and are publicly availableto a person of ordinary skill in the art. For example, a resolutionmodel of the coat protein of Cucumber Green Mottle Mosaic Virus (a coatprotein bearing strong structural similarities to other tobamovirus coatproteins) and the virus can be found in Wang and Stubbs J. Mol. Biol.239:371-384 (1994). Detailed structural information on the virus andcoat protein of Tobacco Mosaic Virus can be found, among other places inNamba et al, J. Mol. Biol. 208:307-325 (1989) and Pattanayek and StubbsJ. Mol. Biol. 228:516-528 (1992).

[0030] Knowledge of the three dimensional structure of a plant virusparticle and the assembly process of the virus particle permits theperson of ordinary skill in the art to design various coat proteinfusion s of the invention, including insertions, and partialsubstitutions. For example, if the protein of interest is of ahydrophilic nature, it may be appropriate to fuse the peptide to theTMVCP region known to be oriented as a surface loop region. Likewise,alpha helical segments that maintain subunit contacts might besubstituted for appropriate regions of the TMVCP helices or nucleic acidbinding domains expressed in the region of the TMVCP oriented towardsthe genome.

[0031] Polynucleotide sequences encoding the subject fusion proteins maycomprise a “leaky” stop codon at a fusion joint. The stop codon may bepresent as the codon immediately adjacent to the fusion joint, or may belocated close (e.g., within 9 bases) to the fusion joint. A leaky stopcodon may be included in polynucleotides encoding the subject coatfusion proteins so as to maintain a desired ratio of fusion protein towild type coat protein. A “leaky” stop codon does not always result intranslational termination and is periodically translated. The frequencyof initiation or termination at a given start/stop codon is contextdependent. The ribosome scans from the 5′-end of a messenger RNA for thefirst ATG codon. If it is in a non-optimal sequence context, theribosome will pass, some fraction of the time, to the next availablestart codon and initiate translation downstream of the first. Similarly,the first termination codon encountered during translation will notfunction 100% of the time if it is in a particular sequence context.Consequently, many naturally occurring proteins are known to exist as apopulation having heterogeneous N and/or C terminal extensions. Thus byincluding a leaky stop codon at a fusion joint coding region in arecombinant viral vector encoding a coat fusion protein, the vector maybe used to produce both a fusion protein and a second smaller protein,e.g., the viral coat protein. A leaky stop codon may be used at, orproximal to, the fusion joints of fusion proteins in which the proteinof interest portion is joined to the carboxyl terminus of the coatprotein region, whereby a single recombinant viral vector may produceboth coat fusion proteins and coat proteins. Additionally, a leaky startcodon may be used at or proximal to the fusion joints of fusion proteinsin which the protein of interest portion is joined to the amino terminusof the coat protein region, whereby a similar result is achieved. In thecase of TMVCP, extensions at the N and C terminus are at the surface ofviral particles and can be expected to project away from the helicalaxis. An example of a leaky stop sequence occurs at the junction of the126/183 kDa reading frames of TMV and was described over 15 years ago(Pelham, H. R. B., 1978). Skuzeski et al. (1991) defined necessary 3′context requirements of this region to confer leakiness of terminationon a heterologous protein marker gene (β-glucuronidase) as CAR-YYA(C=cytidine, A=adenine, Y=pyrimidine).

[0032] In another embodiment of the invention, the fusion joints on thesubject coat fusion proteins are designed so as to comprise an aminoacid sequence that is a substrate for protease. By providing a coatfusion protein having such a fusion joint, the protein of interest maybe conveniently derived from the coat protein fusion by using a suitableproteolytic enzyme. The proteolytic enzyme may contact the fusionprotein either in vitro or in vivo.

[0033] The expression of the subject coat fusion proteins may be drivenby any of a variety of promoters functional in the genome of therecombinant plant viral vector. In a preferred embodiment of theinvention, the subject fusion proteins are expressed from plant viralsubgenomic promoters using vectors as described in U.S. Pat. No.5,316,931.

[0034] Recombinant DNA technologies have allowed the life cycle ofnumerous plant RNA viruses to be extended artificially through a DNAphase that facilitates manipulation of the viral genome. Thesetechniques may be applied by the person ordinary skill in the art inorder make and use recombinant plant viruses of the invention. Theentire cDNA of the TMV genome was cloned and functionally joined to abacterial promoter in an E. coli plasmid (Dawson et al., 1986).Infectious recombinant plant viral RNA transcripts may also be producedusing other well known techniques, for example, with the commerciallyavailable RNA polymerases from T7, T3 or SP6. Precise replicas of thevirion RNA can be produced in vitro with RNA polymerase and dinucleotidecap, m7GpppG. This not only allows manipulation of the viral genome forreverse genetics, but it also allows manipulation of the virus into avector to express foreign genes. A method of producing plant RNA virusvectors based on manipulating RNA fragments with RNA ligase has provedto be impractical and is not widely used (Pelcher, L. E., 1982).Detailed information on how to make and use recombinant RNA plantviruses can be found, among other places in U.S. Pat. No. 5,316,931(Donson et al.), which is herein incorporated by reference. Theinvention provides for polynucleotide encoding recombinant RNA plantvectors for the expression of the subject fusion proteins. The inventionalso provides for polynucleotides comprising a portion or portions ofthe subject vectors. The vectors described in U.S. Pat. No. 5,316,931are particularly preferred for expressing the fusion proteins of theinvention.

[0035] In addition to providing the described viral coat fusionproteins, the invention also provides for virus particles that comprisethe subject fusion proteins. The coat of the virus particles of theinvention may consist entirely of coat fusion protein. In anotherembodiment of the virus particles of the invention, the virus particlecoat may consist of a mixture of coat fusion proteins and non-fusioncoat protein, wherein the ratio of the two proteins may be varied. Astobamovirus coat proteins may self-assemble into virus particles, thevirus particles of the invention may be assembled either in vivo or invitro. The virus particles may also be conveniently dissassembled usingwell known techniques so as to simplify the purification of the subjectfusion proteins, or portions thereof.

[0036] The invention also provides for recombinant plant cellscomprising the subject coat fusion proteins and/or virus particlescomprising the subject coat fusion proteins. These plant cells may beproduced either by infecting plant cells (either in culture or in wholeplants) with infections virus particles of the invention or withpolynucleotides encoding the genomes of the infectious virus particle ofthe invention. The recombinant plant cells of the invention having manyuses. Such uses include serving as a source for the fusion coat proteinsof the invention.

[0037] The protein of interest portion of the subject fusion proteinsmay comprise many different amino acid residue sequences, andaccordingly may different possible biological/chemical propertieshowever, in a preferred embodiment of the invention the protein ofinterest portion of the fusion protein is useful as a vaccine antigen.The surface of TMV particles and other tobamoviruses contain continuousepitopes of high antigenicity and segmental mobility thereby making TMVparticles especially useful in producing a desired immune response.These properties make the virus particles of the invention especiallyuseful as carriers in the presentation of foreign epitopes to mammalianimmune systems.

[0038] While the recombinant RNA viruses of the invention may be used toproduce numerous coat fusion proteins for use as vaccine antigens orvaccine antigen precursors, it is of particular interest to providevaccines against malaria. Human malaria is caused by the protozoanspecies Plasmodium falciparum, P. vivax, P. ovale and P. malariae and istransmitted in the sporozoite form by Anopheles mosquitos. Control ofthis disease will likely require safe and stable vaccines. Severalpeptide epitopes expressed during various stages of the parasite lifecycle are thought to contribute to the induction of protective immunityin partially resistant individuals living in endemic areas and inindividuals experimentally immunized with irradiated sporozoites.

[0039] When the fusion proteins of the invention, portions thereof, orviral particles comprising the fusion proteins are used in vivo, theproteins are typically administered in a composition comprising apharmaceutical carrier. A pharmaceutical carrier can be any compatible,non-toxic substance suitable for delivery of the desired compounds tothe body. Sterile water, alcohol, fats, waxes and inert solids may beincluded in the carrier. Pharmaceutically accepted adjuvants (bufferingagents, dispersing agent) may also be incorporated into thepharmaceutical composition. Additionally, when the subject fusionproteins, or portion thereof, are to be used for the generation of animmune response, protective or otherwise, formulation for administrationmay comprise one or immunological adjuvants in order to stimulate adesired immune response.

[0040] When the fusion proteins of the invention, or portions thereof,are used in vivo, they may be administered to a subject, human oranimal, in a variety of ways. The pharmaceutical compositions may beadministered orally or parenterally, i.e., subcutaneously,intramuscularly or intravenously. Thus, this invention providescompositions for parenteral administration which comprise a solution ofthe fusion protein (or derivative thereof) or a cocktail thereofdissolved in an acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers can be used, e.g., water, buffered water,0.4% saline, 0.3% glycerine and the like. These solutions are sterileand generally free of particulate matter. These compositions may besterilized by conventional, well known sterilization techniques. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, for example sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate, etc. The concentration of fusionprotein (or portion thereof) in these formulations can vary widelydepending on the specific amino acid sequence of the subject proteinsand the desired biological activity, e.g., from less than about 0.5%,usually at or at least about 1% to as much as 15 or 20% by weight andwill be selected primarily based on fluid volumes, viscosities, etc., inaccordance with the particular mode of administration selected.

[0041] Actual methods for preparing parenterally administrablecompositions and adjustments necessary for administration to subjectswill be known or apparent to those skilled in the art and are describedin more detail in, for example, Remington's Pharmaceutical Science,current edition, Mack Publishing Company, Easton, Pa., which isincorporated herein by reference.

[0042] The invention having been described above, may be betterunderstood by reference to the following examples. The examples areoffered by way of illustration and are not intended to be interpreted aslimitations on the scope of the invention.

EXAMPLES

[0043] Biological Deposits

[0044] The following present examples are based on a full length insertof wild type TMV (U1 strain) cloned in the vector pUC18 with a T7promoter sequence at the 5′-end and a KpnI site at the 3′-end (pSNC004,FIG. 2) or a similar plasmid pTMV304. Using the polymerase chainreaction (PCR) technique and primers WD29 (SEQ ID NO: 1) and D1094 (SEQID NO: 2) a 277 XmaI/HindIII amplification product was inserted with the6140 bp XmaI/KpnI fragment from pTMV304 between the KpnI and HindIIIsites of the common cloning vector pUC18 to create pSNC004. The plasmidpTMV304 is available from the American Type Culture Collection,Rockville, Md. (ATCC deposit 45138). The genome of the wild type TMVstrain can be synthesized from pTMV304 using the SP6 polymerase, or frompSNC004 using the T7 polymerase. The wild type TMV strain can also beobtained from the American Type Culture Collection, Rockville, Md. (ATCCdeposit No. PV135). The plasmid pBGC152, Kumagai, M., et al., (1993), isa derivative of pTMV304 and is used only as a cloning intermediate inthe examples described below. The construction of each plasmid vectordescribed in the examples below is diagrammed in FIG. 3.

Example 1

[0045] Propagation and Purification of the U1 Strain of TMV

[0046] The TMVCP fusion vectors described in the following examples arebased on the U1 or wild type TMV strain and are therefore compared tothe parental virus as a control. Nicotiana tabacum cv Xanthi (hereafterreferred to as tobacco) was grown 4-6 weeks after germination, and two4-8 cm expanded leaves were inoculated with a solution of 50 μg/ml TMVU1 by pipetting 100 μl onto carborundum dusted leaves and lightlyabrading the surface with a gloved hand. Six tobacco plants were grownfor 27 days post inoculation accumulating 177 g fresh weight ofharvested leaf biomass not including the two lower inoculated leaves.Purified TMV U1 Sample ID No. TMV204.B4 was recovered (745 mg) at ayield of 4.2 mg of virion per gram of fresh weight by two cycles ofdifferential centrifugation and precipitation with PEG according to themethod of Gooding et al. (1967). Tobacco plants infected with TMV U1accumulated greater than 230 micromoles of coat protein per kilogram ofleaf tissue.

Example 2

[0047] Production of a Malarial B-Cell Epitope Genetically Fused to theSurface Loop Region of the TMVCP

[0048] The monoclonal antibody NVS3 was made by immunizing a mouse withirradiated P. vivax sporozoites. NVS3 mAb passively transferred tomonkeys provided protective immunity to sporozoite infection with thishuman parasite. Using the technique of epitope-scanning with syntheticpeptides, the exact amino acid sequence present on the P. vivaxsporozoite surface and recognized by NVS3 was defined as AGDR (Seq IDNo. P1). The epitope AGDR is contained within a repeating unit of thecircumsporozoite (CS) protein (Charoenvit et al., 1991a), the majorimmunodominant protein coating the sporozoite. Construction of agenetically modified tobamovirus designed to carry this malarial B-cellepitope fused to the surface of virus particles is set forth herein.

[0049] Construction of plasmid pBGC291. The 2.1 kb EcoRI-PstI fragmentfrom pTMV204 described in Dawson, W., et al. (1986) was cloned intopBstSK—(Stratagene Cloning Systems) to form pBGC11. A 0.27 kb fragmentof pBGC11 was PCR amplified using the 5′ primer TB2ClaI5′ (SEQ ID NO: 3)and the 3′ primer CP.ME2+ (SEQ ID NO: 4). The 0.27 kb amplified productwas used as the 5′ primer and C/0AvrII (SEQ ID NO: 5) was the 3′ primerfor PCR amplification. The amplified product was cloned into the SmaIsite of pBstKS+ (Stratagene Cloning Systems) to form pBGC243.

[0050] To eliminate the BstXI and SacII sites from the polylinker,pBGC234 was formed by digesting pBstKS+ (Stratagene Cloning Systems)with BstXI followed by treatment with T4 DNA Polymerase andself-ligation. The 1.3 kb HindIII-KpnI fragment of pBGC304 was clonedinto pBGC234 to form pBGC235. pBGC304 is also named pTMV304 (ATCCdeposit 45138).

[0051] The 0.3 kb PacI-AccI fragment of pBGC243 was cloned into pBGC235to form pBGC244. The 0.02 kb polylinker fragment of pBGC243 (SmaI-EcoRV)was removed to form pBGC280. A 0.02 kb synthetic PstI fragment encodingthe P. vivax AGDR repeat was formed by annealing AGDR3p (SEQ ID NO: 6)with AGDR3m (SEQ ID NO: 7) and the resulting double stranded fragmentwas cloned into pBGC280 to form pBGC282. The 1.0 kb NcoI-KpnI fragmentof pBGC282 was cloned into pSNC004 to form pBGC291.

[0052] The coat protein sequence of the virus TMV291 produced bytranscription of plasmid pBGC291 in vitro is listed in (SEQ ID NO: 16)The epitope (AGDR)3 is calculated to be approximately 6.2% of the weightof the virion.

[0053] Propagation and purification of the epitope expression vector.Infectious transcripts were synthesized from KpnI-linearized pBGC291using T7 RNA polymerase and cap (7mGpppG) according to the manufacturer(New England Biolabs). An increased quantity of recombinant virus wasobtained by passaging and purifying Sample ID No. TMV291.1B1 asdescribed in example 1. Twenty tobacco plants were grown for 29 dayspost inoculation, accumulating 1060 g fresh weight of harvested leafbiomass not including the two lower inoculated leaves. Purified SampleID TMV291.1B2 was recovered (474 mg) at a yield of 0.4 mg virion pergram of fresh weight. Therefore, 25 μg of 12-mer peptide was obtainedper gram of fresh weight extracted. Tobacco plants infected with TMV291accumulated greater than 21 micromoles of peptide per kilogram of leaftissue.

[0054] Product analysis. The conformation of the epitope AGDR containedin the virus TMV291 is specifically recognized by the monoclonalantibody NVS3 in ELISA assays (FIG. 4). By Western blot analysis, NVS3cross-reacted only with the TMV291 cp fusion at 18.6 kD and did notcross-react with the wild type or cp fusion present in TMV261. Thegenomic sequence of the epitope coding region was confirmed by directlysequencing viral RNA extracted from Sample ID No. TMV291.1B2.

Example 3

[0055] Production of a Malarial B-Cell Epitope Genetically Fused to theC Terminus of the TMVCP

[0056] Significant progress has been made in designing effective subunitvaccines using rodent models of malarial disease caused by nonhumanpathogens such as P. yoelii or P. berghei. The monoclonal antibody NYS1recognizes the repeating epitope QGPGAP (SEQ ID NO: 18), present on theCS protein of P. yoelii, and provides a very high level of immunity tosporozoite challenge when passively transferred to mice (Charoenvit, Y.,et al. 1991b). Construction of a genetically modified tobamovirusdesigned to carry this malarial B-cell epitope fused to the surface ofvirus particles is set forth herein.

[0057] Construction of plasmid pBGC261. A 0.5 kb fragment of pBGC11, wasPCR amplified using the 5′ primer TB2ClaI5′ (SEQ ID NO: 3) and the 3′primer C/0AvrII (SEQ ID NO: 5). The amplified product was cloned intothe SmaI site of pBstKS+ (Stratagene Cloning Systems) to form pBGC218.

[0058] pBGC219 was formed by cloning the 0.15 kb AccI-NsiI fragment ofpBGC218 into pBGC235. A 0.05 kb synthetic AvrII fragment was formed byannealing PYCS.1p (SEQ ID NO: 8) with PYCS.1m (SEQ ID NO: 9) and theresulting double stranded fragment, encoding the leaky-stop signal andthe P. yoelii B-cell malarial epitope, was cloned into the AvrII site ofpBGC219 to form pBGC221. The 1.0 kb NcoI-KpnI fragment of pBGC221 wascloned into pBGC152 to form pBGC261.

[0059] The virus TMV261, produced by transcription of plasmid pBGC261 invitro, contains a leaky stop signal at the C terminus of the coatprotein gene and is therefore predicted to synthesize wild type andrecombinant coat proteins at a ratio of 20:1. The recombinant TMVCPfusion synthesized by TMV261 is listed in (SEQ ID NO: 19) with the stopcodon decoded as the amino acid Y (amino acid residue 160). The wildtype sequence, synthesized by the same virus, is listed in (SEQ ID NO:21). The epitope (QGPGAP)2 is calculated to be present at 0.3% of theweight of the virion.

[0060] Propagation and purification of the epitope expression vector.Infectious transcripts were synthesized from KpnI-linearized pBGC261using SP6 RNA polymerase and cap (7mGpppG) according to the manufacturer(Gibco/BRL Life Technologies).

[0061] An increased quantity of recombinant virus was obtained bypassaging and purifying Sample ID No. TMV261.B1b as described inexample 1. Six tobacco plants were grown for 27 days post inoculation,accumulating 205 g fresh weight of harvested leaf biomass not includingthe two lower inoculated leaves. Purified Sample ID No. TMV261.1B2 wasrecovered (252 mg) at a yield of 1.2 mg virion per gram of fresh weight.Therefore, 4 μg of 12-mer peptide was obtained per gram of fresh weightextracted. Tobacco plants infected with TMV261 accumulated greater than3.9 micromoles of peptide per kilogram of leaf tissue.

[0062] Product analysis. The content of the epitope QGPGAP in the virusTMV261 was determined by ELISA with monoclonal antibody NYS1 (FIG. 5).From the titration curve, 50 ug/ml of TMV261 gave the same O.D. reading(1.0) as 0.2 ug/ml of (QGPGAP)2. The measured value of approximately0.4% of the weight of the virion as epitope is in good agreement withthe calculated value of 0.3%. By Western blot analysis, NYS1cross-reacted only with the TMV261 cp fusion at 19 kD and did notcross-react with the wild type cp or cp fusion present in TMV291. Thegenomic sequence of the epitope coding region was confirmed by directlysequencing viral RNA extracted from Sample ID. No. TMV261.1B2.

Example 4

[0063] Production of a Malarial CTL Epitope Genetically Fused to the CTerminus of the TMVCP

[0064] Malarial immunity induced in mice by irradiated sporozoites of P.yoelii is also dependent on CD8+ T lymphocytes. Clone B is one cytotoxicT lymphocyte (CTL) cell clone shown to recognize an epitope present inboth the P. yoelii and P. berghei CS proteins. Clone B recognizes thefollowing amino acid sequence; SYVPSAEQILEFVKQISSQ (SEQ ID NO: 23) andwhen adoptively transferred to mice protects against infection from bothspecies of malaria sporozoites (Weiss et al., 1992). Construction of agenetically modified tobamovirus designed to carry this malarial CTLepitope fused to the surface of virus particles is set forth herein.

[0065] Construction of plasmid pBGC289. A 0.5 kb fragment of pBGC11 wasPCR amplified using the 5′ primer TB2ClaI5′ (SEQ ID NO: 3) and the 3′primer C/-5AvrII (SEQ ID NO: 10). The amplified product was cloned intothe SmaI site of pBstKS+ (Stratagene Cloning Systems) to form pBGC214.

[0066] pBGC215 was formed by cloning the 0.15 kb AccI-NsiI fragment ofpBGC214 into pBGC235. The 0.9 kb NcoI-KpnI fragment from pBGC215 wascloned into pBGC152 to form pBGC216.

[0067] A 0.07 kb synthetic fragment was formed by annealing PYCS.2p (SEQID NO: 11) with PYCS.2m (SEQ ID NO: 12) and the resulting doublestranded fragment, encoding the P. yoelii CTL malarial epitope, wascloned into the AvrII site of pBGC215 made blunt ended by treatment withmung bean nuclease and creating a unique AatII site, to form pBGC262. A0.03 kb synthetic AatII fragment was formed by annealing TLS.1EXP (SEQID NO: 13) with TLS.1EXM (SEQ ID NO: 14) and the resulting doublestranded fragment, encoding the leaky-stop sequence and a stuffersequence used to facilitate cloning, was cloned into AatII digestedpBGC262 to form pBGC263. pBGC262 was digested with AatII and ligated toitself removing the 0.02 kb stuffer fragment to form pBGC264. The 1.0 kbNcoI-KpnI fragment of pBGC264 was cloned into pSNC004 to form pBGC289.

[0068] The virus TMV289 produced by transcription of plasmid pBGC289 invitro, contains a leaky stop signal resulting in the removal of fouramino acids from the C terminus of the wild type TMV coat protein geneand is therefore predicted to synthesize a truncated coat protein and acoat protein with a CTL epitope fused at the C terminus at a ratio of20:1. The recombinant TMVCP/CTL epitope fusion present in TMV289 islisted in SEQ ID NO: 25 with the stop codon decoded as the amino acid Y(amino acid residue 156). The wild type sequence minus four amino acidsfrom the C terminus is listed in SEQ ID NO: 26. The amino acid sequenceof the coat protein of virus TMV216 produced by transcription of theplasmid pBGC216 in vitro, is also truncated by four amino acids. Theepitope SYVPSAEQILEFVKQISSQ (SEQ ID NO:23) is calculated to be presentat approximately 0.5% of the weight of the virion using the sameassumptions confirmed by quantitative ELISA analysis of the readthroughproperties of TMV261 in example 3.

[0069] Propagation and purification of the epitope expression vector.Infectious transcripts were synthesized from KpnI-linearized pBGC289using T7 RNA polymerase and cap (7mGpppG) according to the manufacturer(New England Biolabs). An increased quantity of recombinant virus wasobtained by passaging Sample ID No. TMV289.11B1a as described inexample 1. Fifteen tobacco plants were grown for 33 days postinoculation accumulating 595 g fresh weight of harvested leaf biomassnot including the two lower inoculated leaves. Purified Sample ID. No.TMV289.11B2 was recovered (383 mg) at a yield of 0.6 mg virion per gramof fresh weight. Therefore, 3 μg of 19-mer peptide was obtained per gramof fresh weight extracted. Tobacco plants infected with TMV289accumulated greater than 1.4 micromoles of peptide per kilogram of leaftissue.

[0070] Product analysis. Partial confirmation of the sequence of theepitope coding region of TMV289 was obtained by restriction digestionanalysis of PCR amplified cDNA using viral RNA isolated from Sample ID.No. TMV289.11B2. The presence of proteins in TMV289 with the predictedmobility of the cp fusion at 20 kD and the truncated cp at 17.1 kD wasconfirmed by denaturing polyacrylamide gel electrophoresis.

LITERATURE CITED

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[0075] Charoenvit, Y., Mellouk, S., Cole, C., Bechara, R., Leef, M. F.,Sedegah, M., Yuan, L., Robey, F. A., Beaudoin, R. L., and Hoffman, S. L.1991b. Monoclonal, but not polyclonal, antibodies protect againstPlasmodium yoelii sporozoites. J. Immunol. 146:1020-1025.

[0076] Dawson, W. O., Beck, D. L., Knorr, D. A., and Grantham, G. L.1986. cDNA cloning of the complete genome of tobacco mosaic virus andproduction of infectious transcripts. Proc. Natl. Acad. Sci. USA83:1832-1836.

[0077] Dawson, W. O., Bubrick, P., and Grantham, G. L. 1988.Modifications of the tobacco mosaic virus coat protein gene affectingreplication, movement, and symptomatology. Phytopathology 78:783-789.

[0078] Dawson, W. O., Lewandowski, D. J., Hilf, M. E., Bubrick, P.,Raffo, A. J., Shaw, J. J., Grantham, G. L., and Desjardins, P. R. 1989.A tobacco mosaic virus-hybrid expresses and loses an added gene.Virology 172:285-292.

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[0080] Donson, J., Dawson, W. O., Grantham, G. L., Turpen, T. H.,Turpen, A. M., Garger, S. J., and Grill, L. K. 1992. Recombinant viralvectors having heterologous subgenomic promoters for systemic expressionof foreign genes. U.S. Patent Appl. Serial No. 923,692.

[0081] French, R., Janda, M., and Ahlquist, P. 1986. Bacterial geneinserted in an engineered RNA virus: Efficient expression inmonocotyledonous plant cells. Science 231:1294-1297.

[0082] Gibbs, A. J. 1977. Tobamovirus group, C.M.I./A.A.B. Descriptionsof plant viruses, No. 184. Wm. Culross and Son Ltd., Coupar Angus,Perthshire, Scotland.

[0083] Goelet, P., Lomonossoff, G. P., Butler P. J. G., Akam, M. E., andKarn, J. 1982. Nucleotide sequence of tobacco mosaic virus RNA. Proc.Natl. Acad. Sci. USA 79:5818-5822.

[0084] Gooding, Jr., G. V., and Hebert, T. T. 1967. A simple techniquefor purification of tobacco mosaic virus in large quantities.Phytopathology 57:1285.

[0085] Hamamoto, H., Hashida, E., Matsunaga, Y., Nakagawa, N.,Nakanishi, N., Okada, Y., Sugiyama, Y., and Tsuchimoto, S. 1993a. Plantvirus vector for foreign gene expression—contains foreign gene downstream of viral coat protein gene, linked by read-through sequence. PCTPatent Application WO 93/JP408.

[0086] Hamamoto, H., Sugiyama, Y., Nakagawa, N., Hashida, E., Matsunaga,Y., Takemoto, S., Watanabe Y., and Okada, Y. 1993b. A new tobacco mosaicvirus vector and its use for the systemic production ofangiotensin-I-converting enzyme inhibitor in transgenic tobacco andtomato. Bio/Technology 11:930-932.

[0087] Haynes, J. R., Cunningham, J., von Seefried, A., Lennick, M.,Garvin, R. T., and Shen, S.-H. 1986. Development of agenetically-engineered, candidate polio vaccine employing theself-assembling properties of the tobacco mosaic virus coat protein.Bio/Technology 4:637-641.

[0088] James, E. A., Garvin, R. T., and Haynes, J. R. 1985.Multispecific immunogenic proteins. European Patent Application,174,759.

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[0090] Kumagai, M. H., Turpen, T. H., Weinzettl, N., della-Cioppa, G.,Turpen, A. M., Donson, J., Hilf, M. E., Grantham, G. L., Dawson, W. O.,Chow, T. P., Piatak Jr., M., and Grill, L. K. 1993. Rapid, high levelexpression of biologically active α-trichosanthin in transfected plantsby a novel RNA viral vector. Proc. Natl. Acad. Sci. USA 90:427-430.

[0091] Lomonossoff, G. P., and Johnson, J. E. 1992. Modified plantviruses as vectors. PCT Application WO 92/18618.

[0092] Mason, H. S., Lam, D. M-K., and Arntzen, C. J. 1992. Expressionof hepatitis B surface antigen in transgenic plants. Proc. Natl. Acad.Sci. USA 89:11745-11749.

[0093] Okada, Y., and Han, K. 1986. Plant virus RNA vector. JapanesePatent Application 61/158443.

[0094] Okada, Y., and Takamatsu, N. 1988. A plant virus RNA vector.Japanese Patent Application 63/200789.

[0095] Pelcher, L. E., Halasa, M. C. 1982. An RNA plant virus vector orportion thereof, a method of construction thereof, and a method ofproducing a gene derived product therefrom. European Patent Appl.067,553.

[0096] Pelham, H. R. B. 1978. Leaky UAG termination codon in tobaccomosaic virus RNA. Nature 272:469-471.

[0097] Skuzeski, J. M., Nichols, L. M., Gesteland, R. F., and Atkins, J.F. 1991. The signal for a leaky UAG stop codon in several plant virusesincludes the two downstream codons. J. Mol. Biol. 218:365-373.

[0098] Takamatsu, N., Ishikawa, M., Meshi, T., and Okada, Y. 1987.Expression of bacterial chloramphenicol acetyltransferase gene intobacco plants mediated by TMV-RNA. EMBO J. 6:307-311.

[0099] Takamatsu, N., Watanabe, Y., Yanagi, H., Meshi, T., Shiba, T.,and Okada, Y. 1990. Production of enkephalin in tobacco protoplastsusing tobacco mosaic virus RNA vector. FEBS Lett. 269:73-76.

[0100] Turpen, T. H., and Grill, L. K. Apr. 4, 1989. New productsthrough viral coat protein modification. Biosource Genetics Corporation,Record of Invention, First Written Disclosure.

[0101] Usha, R., Rohll, J. B., Spall, V. E., Shanks, M., Maule, A. J.,Johnson, J. E., and Lomonossoff, G. P. 1993. Expression of an animalvirus antigenic site on the surface of a plant virus particle. Virology197:366-374.

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[0104] Zaitlin, M., and Israel, H. W. 1975. Tobacco mosaic virus (typestrain), C.M.I./A.A.B. Descriptions of plant viruses, No. 151. Wm.Culross and Son Ltd., Coupar Angus, Perthshire, Scotland.

[0105] Incorporation by Reference

[0106] All patents, patents applications, and publications cited areincorporated herein by reference.

[0107] Equivalents

[0108] The foregoing written specification is considered to besufficient to enable one skilled in the art to practice the invention.Indeed, various modifications of the above-described makes for carryingout the invention which are obvious to those skilled in the field ofmolecular biology or related fields are intended to be within the scopeof the following claims.

1 27 49 base pairs nucleic acid unknown unknown DNA (genomic) 1GGAATTCAAG CTTAATACGA CTCACTATAG TATTTTTACA ACAATTACC 49 18 base pairsnucleic acid unknown unknown DNA (genomic) 2 CCTTCATGTA AACCTCTC 18 25base pairs nucleic acid unknown unknown DNA (genomic) 3 TAATCGATGATGATTCGGAG GCTAC 25 36 base pairs nucleic acid unknown unknown DNA(genomic) 4 AAAGTCTCTG TCTCCTGCAG GGAACCTAAC AGTTAC 36 36 base pairsnucleic acid unknown unknown DNA (genomic) 5 ATTATGCATC TTGACTACCTAGGTTGCAGG ACCAGA 36 24 base pairs nucleic acid unknown unknown DNA(genomic) 6 GGCGATCGGG CTGGTGACCG TGCA 24 24 base pairs nucleic acidunknown unknown DNA (genomic) 7 CGGTCACCAG CCCGATCGCC TGCA 24 45 basepairs nucleic acid unknown unknown DNA (genomic) 8 CTAGCAATTA CAAGGTCCAGGTGCACCTCA AGGTCCTGGA GCTCC 45 45 base pairs nucleic acid unknownunknown DNA (genomic) 9 CTAGGGAGCT CCAGGACCTT GAGGTGCACC TGGACCTTGTAATTG 45 35 base pairs nucleic acid unknown unknown DNA (genomic) 10ATTATGCATC TTGACTACCT AGGTCCAAAC CAAAC 35 66 base pairs nucleic acidunknown unknown DNA (genomic) 11 GTCATATGTT CCATCTGCAG AGCAGATCTTGGAATTCGTT AAGCAAATCT CGAGTCAGTA 60 ACTATA 66 66 base pairs nucleic acidunknown unknown DNA (genomic) 12 TATAGTTACT GACTCGAGAT TTGCTTAACGAATTCCAAGA TCTGCTCTGC AGATGGAACA 60 TATGAC 66 33 base pairs nucleic acidunknown unknown DNA (genomic) 13 CGACCTAGGT GATGACGTCA TAGCAATTAA CGT 3333 base pairs nucleic acid unknown unknown DNA (genomic) 14 TAATTGCTATGACGTCATCA CCTAGGTCGA CGT 33 4 amino acids amino acid unknown unknownpeptide 15 Ala Gly Asp Arg 1 510 base pairs nucleic acid unknown unknownDNA (genomic) pBGC291 Fusion CDS 1..510 16 ATG TCT TAC AGT ATC ACT ACTCCA TCT CAG TTC GTG TTC TTG TCA TCA 48 Met Ser Tyr Ser Ile Thr Thr ProSer Gln Phe Val Phe Leu Ser Ser 1 5 10 15 GCG TGG GCC GAC CCA ATA GAGTTA ATT AAT TTA TGT ACT AAT GCC TTA 96 Ala Trp Ala Asp Pro Ile Glu LeuIle Asn Leu Cys Thr Asn Ala Leu 20 25 30 GGA AAT CAG TTT CAA ACA CAA CAAGCT CGA ACT GTC GTT CAA AGA CAA 144 Gly Asn Gln Phe Gln Thr Gln Gln AlaArg Thr Val Val Gln Arg Gln 35 40 45 TTC AGT GAG GTG TGG AAA CCT TCA CCACAA GTA ACT GTT AGG TTC CCT 192 Phe Ser Glu Val Trp Lys Pro Ser Pro GlnVal Thr Val Arg Phe Pro 50 55 60 GCA GGC GAT CGG GCT GGT GAC CGT GCA GGAGAC AGA GAC TTT AAG GTG 240 Ala Gly Asp Arg Ala Gly Asp Arg Ala Gly AspArg Asp Phe Lys Val 65 70 75 80 TAC AGG TAC AAT GCG GTA TTA GAC CCG CTAGTC ACA GCA CTG TTA GGT 288 Tyr Arg Tyr Asn Ala Val Leu Asp Pro Leu ValThr Ala Leu Leu Gly 85 90 95 GCA TTC GAC ACT AGA AAT AGA ATA ATA GAA GTTGAA AAT CAG GCG AAC 336 Ala Phe Asp Thr Arg Asn Arg Ile Ile Glu Val GluAsn Gln Ala Asn 100 105 110 CCC ACG ACT GCC GAA ACG TTA GAT GCT ACT CGTAGA GTA GAC GAC GCA 384 Pro Thr Thr Ala Glu Thr Leu Asp Ala Thr Arg ArgVal Asp Asp Ala 115 120 125 ACG GTG GCC ATA AGG AGC GCG ATA AAT AAT TTAATA GTA GAA TTG ATC 432 Thr Val Ala Ile Arg Ser Ala Ile Asn Asn Leu IleVal Glu Leu Ile 130 135 140 AGA GGA ACC GGA TCT TAT AAT CGG AGC TCT TTCGAG AGC TCT TCT GGT 480 Arg Gly Thr Gly Ser Tyr Asn Arg Ser Ser Phe GluSer Ser Ser Gly 145 150 155 160 TTG GTT TGG ACC TCT GGT CCT GCA ACT TGA510 Leu Val Trp Thr Ser Gly Pro Ala Thr 165 170 169 amino acids aminoacid linear protein pBGC291 Fusion 17 Met Ser Tyr Ser Ile Thr Thr ProSer Gln Phe Val Phe Leu Ser Ser 1 5 10 15 Ala Trp Ala Asp Pro Ile GluLeu Ile Asn Leu Cys Thr Asn Ala Leu 20 25 30 Gly Asn Gln Phe Gln Thr GlnGln Ala Arg Thr Val Val Gln Arg Gln 35 40 45 Phe Ser Glu Val Trp Lys ProSer Pro Gln Val Thr Val Arg Phe Pro 50 55 60 Ala Gly Asp Arg Ala Gly AspArg Ala Gly Asp Arg Asp Phe Lys Val 65 70 75 80 Tyr Arg Tyr Asn Ala ValLeu Asp Pro Leu Val Thr Ala Leu Leu Gly 85 90 95 Ala Phe Asp Thr Arg AsnArg Ile Ile Glu Val Glu Asn Gln Ala Asn 100 105 110 Pro Thr Thr Ala GluThr Leu Asp Ala Thr Arg Arg Val Asp Asp Ala 115 120 125 Thr Val Ala IleArg Ser Ala Ile Asn Asn Leu Ile Val Glu Leu Ile 130 135 140 Arg Gly ThrGly Ser Tyr Asn Arg Ser Ser Phe Glu Ser Ser Ser Gly 145 150 155 160 LeuVal Trp Thr Ser Gly Pro Ala Thr 165 6 amino acids amino acid unknownunknown peptide 18 Gln Gly Pro Gly Ala Pro 1 5 525 base pairs nucleicacid unknown unknown DNA (genomic) pBGC261 Leaky Stop CDS 1..525 19 ATGTCT TAC AGT ATC ACT ACT CCA TCT CAG TTC GTG TTC TTG TCA TCA 48 Met SerTyr Ser Ile Thr Thr Pro Ser Gln Phe Val Phe Leu Ser Ser 1 5 10 15 GCGTGG GCC GAC CCA ATA GAG TTA ATT AAT TTA TGT ACT AAT GCC TTA 96 Ala TrpAla Asp Pro Ile Glu Leu Ile Asn Leu Cys Thr Asn Ala Leu 20 25 30 GGA AATCAG TTT CAA ACA CAA CAA GCT CGA ACT GTC GTT CAA AGA CAA 144 Gly Asn GlnPhe Gln Thr Gln Gln Ala Arg Thr Val Val Gln Arg Gln 35 40 45 TTC AGT GAGGTG TGG AAA CCT TCA CCA CAA GTA ACT GTT AGG TTC CCT 192 Phe Ser Glu ValTrp Lys Pro Ser Pro Gln Val Thr Val Arg Phe Pro 50 55 60 GAC AGT GAC TTTAAG GTG TAC AGG TAC AAT GCG GTA TTA GAC CCG CTA 240 Asp Ser Asp Phe LysVal Tyr Arg Tyr Asn Ala Val Leu Asp Pro Leu 65 70 75 80 GTC ACA GCA CTGTTA GGT GCA TTC GAC ACT AGA AAT AGA ATA ATA GAA 288 Val Thr Ala Leu LeuGly Ala Phe Asp Thr Arg Asn Arg Ile Ile Glu 85 90 95 GTT GAA AAT CAG GCGAAC CCC ACG ACT GCC GAA ACG TTA GAT GCT ACT 336 Val Glu Asn Gln Ala AsnPro Thr Thr Ala Glu Thr Leu Asp Ala Thr 100 105 110 CGT AGA GTA GAC GACGCA ACG GTG GCC ATA AGG AGC GCG ATA AAT AAT 384 Arg Arg Val Asp Asp AlaThr Val Ala Ile Arg Ser Ala Ile Asn Asn 115 120 125 TTA ATA GTA GAA TTGATC AGA GGA ACC GGA TCT TAT AAT CGG AGC TCT 432 Leu Ile Val Glu Leu IleArg Gly Thr Gly Ser Tyr Asn Arg Ser Ser 130 135 140 TTC GAG AGC TCT TCTGGT TTG GTT TGG ACC TCT GGT CCT GCA ACC TAG 480 Phe Glu Ser Ser Ser GlyLeu Val Trp Thr Ser Gly Pro Ala Thr Tyr 145 150 155 160 CAA TTA CAA GGTCCA GGT GCA CCT CAA GGT CCT GGA GCT CCC TAG 525 Gln Leu Gln Gly Pro GlyAla Pro Gln Gly Pro Gly Ala Pro 165 170 175 174 amino acids amino acidlinear protein pBGC261 Leaky Stop 20 Met Ser Tyr Ser Ile Thr Thr Pro SerGln Phe Val Phe Leu Ser Ser 1 5 10 15 Ala Trp Ala Asp Pro Ile Glu LeuIle Asn Leu Cys Thr Asn Ala Leu 20 25 30 Gly Asn Gln Phe Gln Thr Gln GlnAla Arg Thr Val Val Gln Arg Gln 35 40 45 Phe Ser Glu Val Trp Lys Pro SerPro Gln Val Thr Val Arg Phe Pro 50 55 60 Asp Ser Asp Phe Lys Val Tyr ArgTyr Asn Ala Val Leu Asp Pro Leu 65 70 75 80 Val Thr Ala Leu Leu Gly AlaPhe Asp Thr Arg Asn Arg Ile Ile Glu 85 90 95 Val Glu Asn Gln Ala Asn ProThr Thr Ala Glu Thr Leu Asp Ala Thr 100 105 110 Arg Arg Val Asp Asp AlaThr Val Ala Ile Arg Ser Ala Ile Asn Asn 115 120 125 Leu Ile Val Glu LeuIle Arg Gly Thr Gly Ser Tyr Asn Arg Ser Ser 130 135 140 Phe Glu Ser SerSer Gly Leu Val Trp Thr Ser Gly Pro Ala Thr Tyr 145 150 155 160 Gln LeuGln Gly Pro Gly Ala Pro Gln Gly Pro Gly Ala Pro 165 170 480 base pairsnucleic acid unknown unknown DNA (genomic) pBGC261 Nonfusion CDS 1..48021 ATG TCT TAC AGT ATC ACT ACT CCA TCT CAG TTC GTG TTC TTG TCA TCA 48Met Ser Tyr Ser Ile Thr Thr Pro Ser Gln Phe Val Phe Leu Ser Ser 1 5 1015 GCG TGG GCC GAC CCA ATA GAG TTA ATT AAT TTA TGT ACT AAT GCC TTA 96Ala Trp Ala Asp Pro Ile Glu Leu Ile Asn Leu Cys Thr Asn Ala Leu 20 25 30GGA AAT CAG TTT CAA ACA CAA CAA GCT CGA ACT GTC GTT CAA AGA CAA 144 GlyAsn Gln Phe Gln Thr Gln Gln Ala Arg Thr Val Val Gln Arg Gln 35 40 45 TTCAGT GAG GTG TGG AAA CCT TCA CCA CAA GTA ACT GTT AGG TTC CCT 192 Phe SerGlu Val Trp Lys Pro Ser Pro Gln Val Thr Val Arg Phe Pro 50 55 60 GAC AGTGAC TTT AAG GTG TAC AGG TAC AAT GCG GTA TTA GAC CCG CTA 240 Asp Ser AspPhe Lys Val Tyr Arg Tyr Asn Ala Val Leu Asp Pro Leu 65 70 75 80 GTC ACAGCA CTG TTA GGT GCA TTC GAC ACT AGA AAT AGA ATA ATA GAA 288 Val Thr AlaLeu Leu Gly Ala Phe Asp Thr Arg Asn Arg Ile Ile Glu 85 90 95 GTT GAA AATCAG GCG AAC CCC ACG ACT GCC GAA ACG TTA GAT GCT ACT 336 Val Glu Asn GlnAla Asn Pro Thr Thr Ala Glu Thr Leu Asp Ala Thr 100 105 110 CGT AGA GTAGAC GAC GCA ACG GTG GCC ATA AGG AGC GCG ATA AAT AAT 384 Arg Arg Val AspAsp Ala Thr Val Ala Ile Arg Ser Ala Ile Asn Asn 115 120 125 TTA ATA GTAGAA TTG ATC AGA GGA ACC GGA TCT TAT AAT CGG AGC TCT 432 Leu Ile Val GluLeu Ile Arg Gly Thr Gly Ser Tyr Asn Arg Ser Ser 130 135 140 TTC GAG AGCTCT TCT GGT TTG GTT TGG ACC TCT GGT CCT GCA ACC TAG 480 Phe Glu Ser SerSer Gly Leu Val Trp Thr Ser Gly Pro Ala Thr 145 150 155 160 159 aminoacids amino acid linear protein pBGC261 Nonfusion 22 Met Ser Tyr Ser IleThr Thr Pro Ser Gln Phe Val Phe Leu Ser Ser 1 5 10 15 Ala Trp Ala AspPro Ile Glu Leu Ile Asn Leu Cys Thr Asn Ala Leu 20 25 30 Gly Asn Gln PheGln Thr Gln Gln Ala Arg Thr Val Val Gln Arg Gln 35 40 45 Phe Ser Glu ValTrp Lys Pro Ser Pro Gln Val Thr Val Arg Phe Pro 50 55 60 Asp Ser Asp PheLys Val Tyr Arg Tyr Asn Ala Val Leu Asp Pro Leu 65 70 75 80 Val Thr AlaLeu Leu Gly Ala Phe Asp Thr Arg Asn Arg Ile Ile Glu 85 90 95 Val Glu AsnGln Ala Asn Pro Thr Thr Ala Glu Thr Leu Asp Ala Thr 100 105 110 Arg ArgVal Asp Asp Ala Thr Val Ala Ile Arg Ser Ala Ile Asn Asn 115 120 125 LeuIle Val Glu Leu Ile Arg Gly Thr Gly Ser Tyr Asn Arg Ser Ser 130 135 140Phe Glu Ser Ser Ser Gly Leu Val Trp Thr Ser Gly Pro Ala Thr 145 150 15519 amino acids amino acid unknown unknown DNA (genomic) 23 Ser Tyr ValPro Ser Ala Glu Gln Ile Leu Glu Phe Val Lys Gln Ile 1 5 10 15 Ser SerGln 537 base pairs nucleic acid unknown unknown DNA (genomic) pBGC289Leaky Stop CDS 1..537 24 ATG TCT TAC AGT ATC ACT ACT CCA TCT CAG TTC GTGTTC TTG TCA TCA 48 Met Ser Tyr Ser Ile Thr Thr Pro Ser Gln Phe Val PheLeu Ser Ser 1 5 10 15 GCG TGG GCC GAC CCA ATA GAG TTA ATT AAT TTA TGTACT AAT GCC TTA 96 Ala Trp Ala Asp Pro Ile Glu Leu Ile Asn Leu Cys ThrAsn Ala Leu 20 25 30 GGA AAT CAG TTT CAA ACA CAA CAA GCT CGA ACT GTC GTTCAA AGA CAA 144 Gly Asn Gln Phe Gln Thr Gln Gln Ala Arg Thr Val Val GlnArg Gln 35 40 45 TTC AGT GAG GTG TGG AAA CCT TCA CCA CAA GTA ACT GTT AGGTTC CCT 192 Phe Ser Glu Val Trp Lys Pro Ser Pro Gln Val Thr Val Arg PhePro 50 55 60 GAC AGT GAC TTT AAG GTG TAC AGG TAC AAT GCG GTA TTA GAC CCGCTA 240 Asp Ser Asp Phe Lys Val Tyr Arg Tyr Asn Ala Val Leu Asp Pro Leu65 70 75 80 GTC ACA GCA CTG TTA GGT GCA TTC GAC ACT AGA AAT AGA ATA ATAGAA 288 Val Thr Ala Leu Leu Gly Ala Phe Asp Thr Arg Asn Arg Ile Ile Glu85 90 95 GTT GAA AAT CAG GCG AAC CCC ACG ACT GCC GAA ACG TTA GAT GCT ACT336 Val Glu Asn Gln Ala Asn Pro Thr Thr Ala Glu Thr Leu Asp Ala Thr 100105 110 CGT AGA GTA GAC GAC GCA ACG GTG GCC ATA AGG AGC GCG ATA AAT AAT384 Arg Arg Val Asp Asp Ala Thr Val Ala Ile Arg Ser Ala Ile Asn Asn 115120 125 TTA ATA GTA GAA TTG ATC AGA GGA ACC GGA TCT TAT AAT CGG AGC TCT432 Leu Ile Val Glu Leu Ile Arg Gly Thr Gly Ser Tyr Asn Arg Ser Ser 130135 140 TTC GAG AGC TCT TCT GGT TTG GTT TGG ACG TCA TAG CAA TTA ACG TCA480 Phe Glu Ser Ser Ser Gly Leu Val Trp Thr Ser Tyr Gln Leu Thr Ser 145150 155 160 TAT GTT CCA TCT GCA GAG CAG ATC TTG GAA TTC GTT AAG CAA ATCTCG 528 Tyr Val Pro Ser Ala Glu Gln Ile Leu Glu Phe Val Lys Gln Ile Ser165 170 175 AGT CAG TAG 537 Ser Gln 178 amino acids amino acid linearprotein pBGC289 Leaky Stop 25 Met Ser Tyr Ser Ile Thr Thr Pro Ser GlnPhe Val Phe Leu Ser Ser 1 5 10 15 Ala Trp Ala Asp Pro Ile Glu Leu IleAsn Leu Cys Thr Asn Ala Leu 20 25 30 Gly Asn Gln Phe Gln Thr Gln Gln AlaArg Thr Val Val Gln Arg Gln 35 40 45 Phe Ser Glu Val Trp Lys Pro Ser ProGln Val Thr Val Arg Phe Pro 50 55 60 Asp Ser Asp Phe Lys Val Tyr Arg TyrAsn Ala Val Leu Asp Pro Leu 65 70 75 80 Val Thr Ala Leu Leu Gly Ala PheAsp Thr Arg Asn Arg Ile Ile Glu 85 90 95 Val Glu Asn Gln Ala Asn Pro ThrThr Ala Glu Thr Leu Asp Ala Thr 100 105 110 Arg Arg Val Asp Asp Ala ThrVal Ala Ile Arg Ser Ala Ile Asn Asn 115 120 125 Leu Ile Val Glu Leu IleArg Gly Thr Gly Ser Tyr Asn Arg Ser Ser 130 135 140 Phe Glu Ser Ser SerGly Leu Val Trp Thr Ser Tyr Gln Leu Thr Ser 145 150 155 160 Tyr Val ProSer Ala Glu Gln Ile Leu Glu Phe Val Lys Gln Ile Ser 165 170 175 Ser Gln468 base pairs nucleic acid unknown unknown DNA (genomic) pBGC289Non-fusion CDS 1..468 26 ATG TCT TAC AGT ATC ACT ACT CCA TCT CAG TTC GTGTTC TTG TCA TCA 48 Met Ser Tyr Ser Ile Thr Thr Pro Ser Gln Phe Val PheLeu Ser Ser 1 5 10 15 GCG TGG GCC GAC CCA ATA GAG TTA ATT AAT TTA TGTACT AAT GCC TTA 96 Ala Trp Ala Asp Pro Ile Glu Leu Ile Asn Leu Cys ThrAsn Ala Leu 20 25 30 GGA AAT CAG TTT CAA ACA CAA CAA GCT CGA ACT GTC GTTCAA AGA CAA 144 Gly Asn Gln Phe Gln Thr Gln Gln Ala Arg Thr Val Val GlnArg Gln 35 40 45 TTC AGT GAG GTG TGG AAA CCT TCA CCA CAA GTA ACT GTT AGGTTC CCT 192 Phe Ser Glu Val Trp Lys Pro Ser Pro Gln Val Thr Val Arg PhePro 50 55 60 GAC AGT GAC TTT AAG GTG TAC AGG TAC AAT GCG GTA TTA GAC CCGCTA 240 Asp Ser Asp Phe Lys Val Tyr Arg Tyr Asn Ala Val Leu Asp Pro Leu65 70 75 80 GTC ACA GCA CTG TTA GGT GCA TTC GAC ACT AGA AAT AGA ATA ATAGAA 288 Val Thr Ala Leu Leu Gly Ala Phe Asp Thr Arg Asn Arg Ile Ile Glu85 90 95 GTT GAA AAT CAG GCG AAC CCC ACG ACT GCC GAA ACG TTA GAT GCT ACT336 Val Glu Asn Gln Ala Asn Pro Thr Thr Ala Glu Thr Leu Asp Ala Thr 100105 110 CGT AGA GTA GAC GAC GCA ACG GTG GCC ATA AGG AGC GCG ATA AAT AAT384 Arg Arg Val Asp Asp Ala Thr Val Ala Ile Arg Ser Ala Ile Asn Asn 115120 125 TTA ATA GTA GAA TTG ATC AGA GGA ACC GGA TCT TAT AAT CGG AGC TCT432 Leu Ile Val Glu Leu Ile Arg Gly Thr Gly Ser Tyr Asn Arg Ser Ser 130135 140 TTC GAG AGC TCT TCT GGT TTG GTT TGG ACG TCA TAG 468 Phe Glu SerSer Ser Gly Leu Val Trp Thr Ser 145 150 155 155 amino acids amino acidlinear protein pBGC289 Non-fusion 27 Met Ser Tyr Ser Ile Thr Thr Pro SerGln Phe Val Phe Leu Ser Ser 1 5 10 15 Ala Trp Ala Asp Pro Ile Glu LeuIle Asn Leu Cys Thr Asn Ala Leu 20 25 30 Gly Asn Gln Phe Gln Thr Gln GlnAla Arg Thr Val Val Gln Arg Gln 35 40 45 Phe Ser Glu Val Trp Lys Pro SerPro Gln Val Thr Val Arg Phe Pro 50 55 60 Asp Ser Asp Phe Lys Val Tyr ArgTyr Asn Ala Val Leu Asp Pro Leu 65 70 75 80 Val Thr Ala Leu Leu Gly AlaPhe Asp Thr Arg Asn Arg Ile Ile Glu 85 90 95 Val Glu Asn Gln Ala Asn ProThr Thr Ala Glu Thr Leu Asp Ala Thr 100 105 110 Arg Arg Val Asp Asp AlaThr Val Ala Ile Arg Ser Ala Ile Asn Asn 115 120 125 Leu Ile Val Glu LeuIle Arg Gly Thr Gly Ser Tyr Asn Arg Ser Ser 130 135 140 Phe Glu Ser SerSer Gly Leu Val Trp Thr Ser 145 150 155

What is claimed is:
 1. A polynucleotide encoding fusion protein, thefusion protein consisting essentially of a tobamovirus coat proteinfused to a protein of interest at a fusion joint.
 2. A polynucleotideaccording to claim 1, wherein the fusion is an amino terminus fusion. 3.A polynucleotide according to claim 1, wherein the fusion is a carboxyterminus fusion.
 4. A polynucleotide according to claim 1, wherein thefusion is an internal fusion.
 5. A polynucleotide according to claim 1,wherein the fusion joint comprises a leaky stop codon.
 6. Apolynucleotide according to claim 1, wherein the fusion joint comprisesa leaky start codon.
 7. A polynucleotide according to claim 1, whereinthe protein of interest is an antigen.
 8. A polynucleotide according toclaim 1, wherein the coat protein is a tobacco mosaic virus coatprotein.
 9. A recombinant plant viral genome comprising a polynucleotideaccording to claim
 1. 10. A recombinant plant virus particle, comprisinga genome according to claim
 9. 11. A polypeptide encoded by apolynucleotide according to claim
 1. 12. A recombinant plant virus,wherein the coat protein is encoded by a polynucleotide according toclaim
 1. 13. A plant cell comprising a polynucleotide according to claim9.